Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.1 (phosphodiesterase)
18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

I have shown that cyclic AMP stimulates sugar uptake in rat thymocytes. However, trypsin treatment, which increases rat thymocyte cyclic AMP concentration, fails to increase sugar uptake. The purpose of the present study is to examine this seeming inconsistency, and to evaluate further the function of trypsin. Mild trypsin treatment of rat thymocytes produced a dose-related increase in cellular cyclic AMP concentration. Trypsin produced the same proportionate increase in cyclic AMP concentration in the presence or absence of optimal concentrations of the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine, which suggests that trypsin acts to increase thymocyte cyclic AMP concentration by stimulating adenylate cyclase activity. Trypsin at concentrations of 0.3 mg/ml and less had no effect on the uptake of the glucose analogue 2-deoxy-D-glucose (2-DG), whereas at concentrations of 1 mg/ml and higher trypsin produced a small, dose-related, decrease in basal 2-DG uptake, becoming significantly lower than control values only at 5 mg/ml (-22.7%, P less than 0.05). Thymocyte sugar transporters, characterized by means of cytochalasin B binding, consist of a single class of sites with an apparent KD of 0.15 microM and maximum binding capacity of 2.73 pmol/20 x 10(6) cells (8.4 x 10(4) sites/thymocyte). Trypsin produced a dose-related decrease in the sugar-displaceable binding of cytochalasin B, so that at 5 mg of trypsin/ml the number of sugar transporters was decreased by approx. 50%. Thus trypsin treatment of rat thymocytes on the one hand increases cellular cyclic AMP concentration, which itself potentiates 2-DG uptake, and on the other hand decreases the number of sugar transporters, which itself decreases cellular sugar uptake, indicating that the apparent effect of trypsin on thymocyte 2-DG uptake is the result of the balance of its effects on these two systems.
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PMID:The effect of trypsin on sugar uptake in rat thymocytes. Modulation of cellular cyclic AMP concentration and the sugar-transport system. 282 42

Extraction of frozen canine cardiac muscle rendered soluble over 90% of the cyclic AMP phosphodiesterase activity. The residual activity was membrane-bound. Ion exchange chromatography of the soluble activity on DE-52 allowed for the resolution of three distinct cyclic AMP phosphodiesterase fractions termed PDE-I, PDE-II and PDE-III in order of elution from the column by a linear NaCl gradient. The relative ratio of cyclic AMP phosphodiesterase activity exhibited by these three peaks was 1:0.65:0.82 and of cyclic GMP phosphodiesterase activity was 1:0.52:0.05 for PDE-I, PDE-II and PDE-III respectively. PDE-II and PDE-III were further purified by re-chromatography on DE-52. Fractions PDE-II and PDE-III were thermolabile at 50 degrees, decaying as single exponentials with half lives of 180 sec and 77 sec respectively. All three species exhibited non-linear Lineweaver-Burke plots for the hydrolysis of cyclic AMP, exhibiting both high and low affinity components. Hydrolysis of cyclic GMP by all three components obeyed normal kinetics, yielding linear plots. PDE-I was a Ca2+/calmodulin-activated species which exhibited a low Km for both cyclic AMP and cyclic GMP but hydrolysed cyclic GMP with a higher Vmax than for cyclic AMP. PDE-II exhibited a much lower Km for cyclic AMP than for cyclic GMP and a much higher Vmax for the hydrolysis of cyclic AMP. PDE-III exhibited a low Km for both cyclic AMP and cyclic GMP, however, its Vmax for cyclic AMP was about 40-fold higher than for cyclic GMP. Cyclic GMP acted as a potent inhibitor (IC50 = 6.3 microM) of cyclic AMP hydrolysis catalysed by PDE-III but not of the hydrolysis of cyclic AMP by PDE-II (IC50 = 33.2 microM). The phosphodiesterase inhibitors milrinone, CI-930, UK-35,493, carbazeran and buquineran acted as potent inhibitors of cyclic AMP hydrolysis catalysed by both PDE-II and PDE-III enzymes. They did not inhibit PDE-I activity. PDE-II, when prepared in the absence of protease inhibitors exhibited a reduced potency to inhibition by these compounds. Treatment of purified PDE-II with trypsin caused a reduction in enzyme activity and reduced dramatically the sensitivity of PDE-II activity to inhibition by these various compounds. The action of proteolysis in attenuating the inhibitory effect of these compounds on PDE-II was most dramatic with CI-930, milrinone, amrinone, buquineran and UK35,493 and least dramatic with carbazeran and IBMX.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Proteolysis of cyclic AMP phosphodiesterase-II attenuates its ability to be inhibited by compounds which exert positive inotropic actions in cardiac tissue. 282 12

The switching on of the cGMP phosphodiesterase (PDE) in retinal rod outer segments by activated transducin (T alpha-GTP) is a key step in visual excitation. The finding that trypsin activates PDE (alpha beta gamma) by degrading its gamma subunit and the reversal of this activation by gamma led to the proposal that T alpha-GTP activates PDE by relieving an inhibitory constraint imposed by gamma (Hurley and Stryer: J. Biol. Chem. 257:11094-11099, 1982). We report here studies showing that the addition of gamma subunit also reverses the activation of PDE by T alpha-GTP-gamma S. A procedure for preparing gamma in high yield (50-80%) is presented. Analyses of SDS polyacrylamide gel slices confirmed that inhibitory activity resides in the gamma subunit. Nanomolar gamma blocks the activation of PDE by micromolar T alpha-GTP gamma S. The degree of activation of PDE depends reciprocally on the concentrations of gamma and T alpha-GTP gamma S. gamma remains bound to the disk membrane during the activation of PDE by transducin. The binding of gamma to the alpha beta subunits of native PDE is very tight; the dissociation constant is less than 10 pM, indicating that fewer than 1 in 1,700 PDE molecules in rod outer segments are activated in the absence of T alpha-GTP.
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PMID:Reciprocal control of retinal rod cyclic GMP phosphodiesterase by its gamma subunit and transducin. 283 61

Cyclic GMP-specific phosphodiesterase (3',5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 1.3.4.17) (PDE) is thought to be a key enzyme of the retinal-rod phototransduction cyclic nucleotide pathway. We attempted to investigate the properties and content of PDE in retinal-cone photoreceptors. The fractions obtained from cone-dominant ground squirrel retinas were analyzed for cone visual pigment content and PDE activity. The cone visual pigment content was estimated to be approx. 65 pmol per retina. The distribution of cone visual pigment coincided with that of the PDE activity through several steps of photoreceptor membrane purification by sucrose density gradient centrifugation. The ground squirrel retinal PDE was similar to the retinal-rod PDE by its kinetic properties, thermostability, sensitivity to tryptic activation, Stokes radius and pI values. The cone visual pigment enriched fractions contained the heat-stable trypsin-inactivated PDE inhibitor. Its functional properties seem to be similar to those of the retinal-rod PDE inhibitory subunit. The PDE content in ground squirrel retina was roughly estimated to be about five copies of enzyme per 100 cone visual pigment molecules. The obtained results indicated that the major portion of ground squirrel retinal cyclic GMP-specific PDE is the endogenous cone photoreceptor membrane enzyme and strongly supported the conception about the key role of PDE in cone phototransduction. The existence of essential differences between rod and cone systems rapidly returning cyclic GMP-specific amplification cascade components to the dark (or inactivated) states after photon absorption was suggested. If this suggestion is true, the well-known distinctions between response kinetics and light sensitivity of these two kinds of photoreceptor can be explained.
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PMID:Properties and content of cyclic nucleotide phosphodiesterase in photoreceptor outer segments of ground squirrel retina. 283 85

The biochemical bases for the differences in cone and rod photoreceptor physiology have not been thoroughly examined because of the difficulty in obtaining cone photoreceptor components. We report here the purification and preliminary characterization of a bovine cyclic GMP phosphodiesterase (PDE) which is enriched in cone photoreceptors. The cone PDE was purified at least 15,000-fold to apparent homogeneity from bovine retinas by DEAE-cellulose and cGMP-Sepharose affinity chromatography. The trypsin-activated cone PDE hydrolyzed cGMP with efficiency similar to that of the rod PDE. However, a number of characteristics distinguished the cone PDE from the rod isozyme including the subunit structure. As previously reported, the apparent molecular weight of the cone PDE large subunit (alpha') was slightly larger than either of the large subunits of the rod PDE (93,500 versus 88,000 and 84,000). Three other smaller polypeptides were associated with the alpha' subunit (Mr = 11,000, 13,000, and 15,000), one of which (11,000) may be identical to the rod PDE gamma subunit. Cone phosphodiesterase binds at least 10-fold more cyclic GMP/mol of PDE than the rod photoreceptor isozyme. Cyclic GMP binds to this noncatalytic site with high affinity (Kd = 11 nM) and dissociates very slowly (t1/2 = 10-20 min at 37 degrees C). Purified rod transducin activated the cone PDE in solution to at least 90% of the trypsin-activated level. The concentration of rod transducin required for half-maximal activation of cone PDE (15 nM) was 50-fold lower than that necessary for half-maximal activation of rod PDE. Thus several properties of the cone phosphodiesterase clearly distinguish it from the rod isozyme and could account for some differences in cone and rod physiology.
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PMID:Characterization of a bovine cone photoreceptor phosphodiesterase purified by cyclic GMP-sepharose chromatography. 283 13

Pretreatment of an affinity-purified, brain calmodulin (CaM)-dependent phosphodiesterase (EC 3.1.4.17) with p-hydroxyphenylglyoxal (pHPG), a specific arginine-modifying reagent, resulted in a time-dependent loss in CaM-stimulated hydrolysis of cyclic AMP and cyclic GMP with no change in basal, CaM-independent activity. The loss in CaM-stimulated activity was preceded by a transient increase in CaM-dependent activity. Phenylglyoxal was 10-fold more effective than pHPG in promoting the loss of CaM-stimulated activity with a second-order rate constant of 13.3 M-1 min-1. Other arginine-modifying reagents, 1,2-cyclohexanedione and 2,3-butanedione, were not effective. The pHPG-modified enzyme was activated by 100 microM lysophosphatidylcholine to levels comparable to CaM-stimulated activity. The arginyl-modified enzyme was also activated by chymotrypsin and trypsin but not to the extent of the untreated enzyme stimulated with CaM. The presence of CaM during chemical modification with pHPG protected the enzyme from inactivation. Both the extent of activation and the amount of CaM necessary for 50% maximal activation were affected by pHPG treatment of the enzyme. The approximate number of modified arginines estimated by [7-14C]phenylglyoxal incorporation and amino acid analysis after complete inactivation of CaM stimulation was seven residues per catalytic subunit assuming enzyme homogeneity. The Stokes radius and sedimentation coefficient of the enzyme were unchanged by the modification. These results suggest that arginine residues are critical for functional interaction between phosphodiesterase and CaM and that controlled modification can selectively alter CaM-stimulated enzyme activity.
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PMID:Involvement of arginine residues in the activation of calmodulin-dependent 3',5'-cyclic-nucleotide phosphodiesterase. 283 86

Membrane-bound low-Km cAMP phosphodiesterase (PDE) was activated when intact rat fat cells were incubated with somatomedin C. Somatomedin C rapidly stimulated the enzyme, reaching a maximum reaction in 5 to 10 minutes. By kinetic analysis, somatomedin C activated PDE by increasing the maximal velocity (Vmax) values without altering the Michaelis-Menten constant (Km) values (0.24 +/- 0.03 mumol/L). The ED50 value of the activation by somatomedin C was very high (38.0 +/- 3.2 nmol/L) compared with that of insulin (0.22 +/- 0.07 nmol/L). This indicates that somatomedin C was about 173 times less potent than insulin in the stimulation of PDE. This potency ratio is similar to those that have been reported on lipid formation or on the other biologic insulinlike activities. When the insulin receptors were destroyed by trypsin treatment, effects of somatomedin C on the enzyme activation were abolished. This finding suggests that activation of PDE by somatomedin C was mediated through the insulin receptor.
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PMID:Effect of somatomedin C on insulin-sensitive phosphodiesterase in rat fat cells. 283 32

The purified catalytic subunit (C) of cAMP-dependent protein kinase produced a 2-fold activation of the low Km phosphodiesterase in crude microsomes (P-2 pellet) of rat adipocytes. This activation was C subunit concentration-dependent, ATP-dependent, blocked by a specific peptide inhibitor, and lost if the C subunit was first heat denatured. The concentration of ATP necessary for half-maximal activation of the low Km phosphodiesterase was 4.50 +/- 1.1 microM, which was nearly the same as the known Km of C subunit for ATP (3.1 microM) using other substrates. The concentration of C subunit producing half-maximal activation of phosphodiesterase was 0.22 +/- 0.04 microM, slightly less than the measured concentration of total C subunit in adipocytes (0.45 microM). The activation of the low Km phosphodiesterase by C subunit was specific, since on an equimolar basis, myosin light chain kinase, cGMP-dependent protein kinase, or Ca2+/calmodulin-dependent protein kinase II did not activate the enzyme. The percent stimulation of phosphodiesterase by C subunit was about the same as that produced by incubation of adipocytes with a cAMP analog, and the enzyme first activated in vivo with the analog was not activated to the same extent (on a percentage basis) by in vitro treatment with C subunit. Treatment of the crude microsomes with trypsin resulted in transfer of phosphodiesterase catalytic activity from the particulate to the supernatant fraction, but the enzyme in the supernatant was minimally activated by C subunit, suggesting either loss or dislocation of the regulatory component. The C subunit-mediated activation of phosphodiesterase was preserved after either transfer of phosphodiesterase activity to the supernatant fraction by nonionic detergents or partial purification of the transferred enzyme. The present findings are consistent with the suggestion that protein kinase regulates the concentration of cAMP through phosphodiesterase activation and provide direct evidence that the mechanism of activation involves phosphorylation.
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PMID:Activation of the particulate low Km phosphodiesterase of adipocytes by addition of cAMP-dependent protein kinase. 283 86

Effect of sera with anti-insulin receptor antibodies (AIRS) on insulin-sensitive phosphodiesterase in rat fat cells was examined. AIRS activated the enzyme when incubated with intact fat cells. AIRS (1:400 dilution) were less potent for activation of the phosphodiesterase than insulin (3 nM), but were more potent for inhibition of 125I-insulin binding to fat cells than insulin. When insulin receptor of fat cells was destroyed with trypsin-treatment, AIRS as well as insulin completely lost the ability to activate the phosphodiesterase. These findings suggest that AIRS bind to or very near the insulin receptor and exhibit insulin-like biological effect of the phosphodiesterase activation.
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PMID:Effect of anti-insulin receptor antibodies on insulin-sensitive phosphodiesterase in rat fat cells. 284 Dec 15

Membrane-associated, Type II (cGMP-activatable) cyclic nucleotide phosphodiesterase (PDE) from rabbit brain, representing 75% of the total homogenate Type II PDE activity, was purified to apparent homogeneity. The enzyme was released from 13,000 x g particulate fractions by limited proteolysis with trypsin and fractionated using DE-52 anion-exchange, cGMP-Sepharose affinity and hydroxylapatite chromatographies. The enzyme showed 105 kDa subunits by SDS-PAGE and had a Stokes radius of 62.70 A as determined by gel filtration chromatography. Hydrolysis of cAMP or cGMP showed positive cooperativity, with cAMP kinetic behavior linearized in the presence of 2 microM cGMP. Substrate concentrations required for half maximum velocity were 28 microM for cAMP and 16 microM for cGMP. Maximum velocities were approx. 160 mumol/min per mg for both nucleotides. The apparent Kact for cGMP stimulation of cAMP hydrolysis at 5 microM substrate was 0.35 microM and maximal stimulation (3-5-fold) was achieved with 2 microM cGMP. Cyclic nucleotide hydrolysis was not enhanced by calcium/calmodulin. The purified enzyme can be labeled by cAMP-dependent protein kinase as demonstrated by the incorporation of 32P from [gamma-32P]ATP into the 105 kDa enzyme subunit. Initial experiments showed that phosphorylation of the enzyme did not significantly alter enzyme activity measured at 5 microM [3H]cAMP in the absence or presence of 2 microM cGMP or at 40 microM [3H]cGMP. Monoclonal antibodies produced against Type II PDE immunoprecipitate enzyme activity, 105 kDa protein and 32P-labeled enzyme. The 105 kDa protein was also photoaffinity labeled with [32P]cGMP. The purified Type II PDE described here is physicochemically very similar to the isozyme purified from the cytosolic fraction of several bovine tissues with the exception that it is predominantly a particulate enzyme. This difference may reflect an important regulatory mechanism governing the metabolism of cyclic nucleotides in the central nervous system.
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PMID:Purification and partial characterization of membrane-associated type II (cGMP-activatable) cyclic nucleotide phosphodiesterase from rabbit brain. 284 74


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